# 2.2.8 研究 | 2021-多组学分析肺结核队列的记忆T细胞状态

## 速览

* 题目：Multimodally profiling memory T cells from a tuberculosis cohort identifies cell state associations with demographics, environment and disease
* 作者：哈佛医学院 Soumya Raychaudhuri 团队

  > 首先感觉他们实验室主页做的很不错：<https://immunogenomics.hms.harvard.edu/lab> ![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-061211.png)
* 链接：<https://www.nature.com/articles/s41590-021-00933-1>
* 发表在：Nature Immunology （2021-05-24）
* 数据是公开的：[GSE158769](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE158769) ，采用CITE-seq

  ```
  Platforms    GPL11154    Illumina HiSeq 2000 (Homo sapiens)
  Samples      GSM4810298    Memory_T_cell_CITE-seq

  GSE158769_exprs_norm.tsv.gz    3.0 Gb    (ftp)(http)    TSV
  GSE158769_exprs_raw.tsv.gz    747.0 Mb    (ftp)(http)    TSV
  GSE158769_meta_data.txt.gz    19.8 Mb    (ftp)(http)    TXT
  ```

  Genotype data：[phs002025.v1.p1](https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs002025.v1.p1)
* 分析代码：<https://github.com/immunogenomics/TB_Tcell_CITEseq> ![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-082342.png)
* 还发开了一个网页：<https://immunogenomics.io/tbru/>

## 摘要

* 分析了来自秘鲁的259个肺结核（tuberculosis , TB)病人的500,089个记忆T细胞，得到31种细胞状态
* 在排除年龄、性别、季节、遗传等因素影响后，发现了一种多能型type 17 helper T (TH17) 细胞状态，在结核杆菌发展成肺结核病的过程中丰度和功能都有所下降

## 方法

**数据准备**

* PBMC sample preparation
* Flow cytometry of total PBMCs
* CITE-seq of isolated memory T cells
* Bulk RNA sequencing
* Genotyping and genetic data processing

**数据分析**

* scRNA比对+定量
* scRNA数据拆分
* scRNA数据质控
* 降维
* 聚类+注释
* bulk RNA-seq 比对+定量
* 利用MASC(modeling of associations of single cells )评价不同细胞状态与疾病的关系

## 先用CITE-seq测了>500,000个T细胞

* 在秘鲁首都利马进行了一次大型的流行病学调查（n= 18,544），从其中招募了264个人，采样了外周血单核细胞进行CITE-seq测定，同时还加入了基因表型数据和蛋白数据
* 图b是进行了6次QC，每一次过滤掉一些细胞，最后得到了500K
* 大部分样本的细胞数量在2k左右

![](https://media.springernature.com/full/springer-static/image/art%3A10.1038%2Fs41590-021-00933-1/MediaObjects/41590_2021_933_Fig1_HTML.png)

## 得到记忆T细胞的31种不同状态

利用canonical correlation analysis (CCA) 进行降维，选择top20 canonical variates (CVs)进行批次校正和聚类，根据基因和蛋白marker得到31个细胞状态

* 23/31 were CD4+; five were CD8+; one (C-24) was a mixture; Two clusters (C-30 and C-31) were CD4−CD8−
* **C-20**： contained a subset of CD4−CD8− and CD8+ cells expressing innate-like T cell markers, including ZBTB16 and CD161 and CD26 surface proteins
* one central memory cluster (**C-25**) and distinct *GZMK*+ (**C-28**) and *GZMB*+ (**C-29**) effector subsets, reflecting different cytotoxicities
* high expression of HLA-DR and CD38 surface protein and proliferation-associated *MKI67* (**C-15** and **C-27**) represent chronically activated cells
* TH17 cells (**C-12**, CCR6+ and *RORC*)&#x20;
* type 1 helper T cells (TH1; **C-17**, CXCR3+ and *IFNG* and *TBX21*)
* heterogeneous continuum of intermediate TH1/TH17 states (**C-13**, **C-16** and **C-19**) with varying degrees of CXCR3, CCR6, CCR5 and CD161 surface protein expression and *RORC* and *TBX21* expression
* CD161+ subset of type 2 helper T (TH2) cells (**C-14**), described as pathogenic, with higher expression of allergy-associated *HPGDS* and *IL17RB*
* a subset of *FOXP3*+ Treg cells (**C-5**) expressing higher CCR6 surface protein and *CTLA4* and *RORC* than other Treg cells

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-070418.png)

## 记忆T细胞的状态因人口特征和环境而异

发现： 年龄因素的影响最大；性别因素与T细胞状态也高度相关； type 2 helper T (TH2) 细胞状态在冬天（季节因素）搜集的样本中最为丰富

![](https://media.springernature.com/full/springer-static/image/art%3A10.1038%2Fs41590-021-00933-1/MediaObjects/41590_2021_933_Fig3_HTML.png)

## 之后发现一个感兴趣的C-12状态

探索每个细胞状态和肺结核病情进展的关系，同时校正其他可能存在影响的因素（年龄、性别、季节、遗传以及其他技术因素），发现：

**病人在肺结核发展时期，C-12显著降低了20%**，并且在年龄因素和男性因素下也会下降，而季节因素（冬季）会上升

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-073932.png)

又找到了**C-12 surface markers：CD4+CD26+CD161+CCR6+** ，这些marker已经被证明了以下功能：

* IL-17-producing TH17 state
* CD26 is a co-stimulatory molecule that promotes cytotoxicity
* CD161 is associated with innate-like function
* CCR6 is a homing marker that directs migration to inflamed sites

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-075041.png)

那么C-12的下降是疾病的原因还是结果呢？

利用外部数据进行验证，发现这种变化可能早于疾病发展

* predicted C-12 abundance was 9% lower in cases before progression compared to latent controls&#x20;

![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-082142.png)


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